Project Summary/Abstract The overall goal of the project is to achieve holistic understanding of structure and function of heteronuclear metalloenzymes involved in multi-electron redox processes, which are more difficult to study than homonuclear enzymes, and to address important scientific issues in the fields of respiration and the global nitrogen and sulfur cycles. Specifically, we seek to investigate why a heme-nonheme Fe center in nitric oxide reductase (NOR) is effective at 2e- reduction of NO, allowing N-N bond formation, whereas a heme-Cu center in heme-Cu oxidase (HCO) is proficient at 4e- reduction of O2, enabling O-O bond cleavage, while both a different heme-Cu center in sulfite reductase from W. succinogenes (SiRA) and a heme-Fe4S4 center in assimilatory sulfite reductases (SiR) are efficient at 6e- reduction of sulfite, promoting S-O bond cleavage. To achieve this goal, the proposal is based on a scientific premise that developing a novel biosynthetic approach using stable, easy-to-produce, and well-characterized heme proteins as scaffolds for making structural and functional models of HCO, NOR, SiRA and SiR can overcome critical methodological barriers to progress in the field. We will use the biosynthetic models to 1) Understand how a heme-Cu center can exhibit either HCO or SiR activity, 2) elucidate structural features responsible for catalytic activity and substrate binding affinity of SiR, 3) clarify the roles of Tyr in HCO and SiR activities, and 4) investigate the roles of heme cofactors in HCO, NOR and SiR activities. To ensure scientific rigor, we will use activity as guidance for our project design and spectroscopic, crystallographic and computational techniques to characterize our models. Achieving the above goals will result in deeper understanding of the structure and function of HCO, NOR, SiRA and SiR that may be very difficult to achieve by studying the native enzymes alone. The ability to place different heteronuclear metal centers into the same protein scaffold offers insight into similarities and differences between the four heteronuclear metalloenzymes. In doing so, the project will advance the knowledge of metalloprotein structure, function, and design in general, as the guiding principles obtained from these studies will be applicable to a broad range of metalloenzymes important for human health.